Each of the over thirty known lysosomal storage diseases (LSDs) is characterized by a similar pathogenesis, namely, a compromised lysosomal hydrolase. Generally, the activity of a single lysosomal hydrolytic enzyme is reduced or lacking altogether, usually due to inheritance of an autosomal recessive mutation. As a consequence, the substrate of the compromised enzyme accumulates undigested in lysosomes, producing severe disruption of cellular architecture and various disease manifestations.
2.1 Lysosomal Storage Diseases
Gaucher's disease, first described by Phillipe C. E. Gaucher in 1882, is the oldest and most common lysosomal storage disease known. Type 1 is the most common among three recognized clinical types and follows a chronic course which does not involve the nervous system. Types 2 and 3 both have a CNS component, the former being an acute infantile form with death by age two and the latter a subacute juvenile form. The incidence of Type 1 Gaucher's disease is about one in 50,000 live births generally and about one in 400 live births among Ashkenazim (see generally Kolodny et al., 1998, “Storage Diseases of the Reticuloendothelial System”, In: Nathan and Oski's Hematology of Infancy and Childhood, 5th ed., vol. 2, David G. Nathan and Stuart H. Orkin, Eds., W. B. Saunders Co., pages 1461-1507). Also known as glucosylceramide lipidosis, Gaucher's disease is caused by inactivation of the enzyme glucocerebrosidase and accumulation of glucocerebroside. Glucocerebrosidase normally catalyzes the hydrolysis of glucocerebroside to glucose and ceramide. In Gaucher's disease, glucocerebroside accumulates in tissue macrophages which become engorged and are typically found in liver, spleen and bone marrow and occasionally in lung, kidney and intestine. Secondary hematologic sequelae include severe anemia and thrombocytopenia in addition to the characteristic progressive hepatosplenomegaly and skeletal complications, including osteonecrosis and osteopenia with secondary pathological fractures.
Fabry disease is an X-linked recessive LSD characterized by a deficiency of α-galactosidase A (α-Gal A), also known as ceramide trihexosidase, which leads to vascular and other disease manifestations via accumulation of glycosphingolipids with terminal α-galactosyl residues, such as globotriaosylceramide (GL-3) (see generally Desnick R J et al., 1995, α-Galactosidase A Deficiency: Fabry Disease, In: The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th ed., pages 2741-2784). Symptoms may include anhidrosis (absence of sweating), painful fingers, left ventricular hypertrophy, renal manifestations, and ischemic strokes. The severity of symptoms varies dramatically (Grewal R P, 1994, Stroke in Fabry's Disease, J. Neurol. 241, 153-156). A variant with manifestations limited to the heart is recognized, and its incidence may be more prevalent than once believed (Nakao S, 1995, An Atypical Variant of Fabry's Disease in Men with Left Ventricular Hypertrophy, N. Engl. J. Med. 333, 288-293). Recognition of unusual variants can be delayed until quite late in life, although diagnosis in childhood is possible with clinical vigilance (Ko Y H et al., 1996, Atypical Fabry's Disease—An Oligosymptomatic Variant, Arch. Pathol. Lab. Med. 120, 86-89; Mendez M F et al., 1997, The Vascular Dementia of Fabry's Disease, Dement. Geriatr. Cogn. Disord. 8, 252-257; Shelley E D et al., 1995, Painful Fingers, Heat Intolerance, and Telangiectases of the Ear: Easily Ignored Childhood Signs of Fabry Disease, Pediatric Derm. 12, 215-219). The mean age of diagnosis of Fabry disease is 29 years.
Niemann-Pick disease, also known as sphingomyelin lipidosis, comprises a group of disorders characterized by foam cell infiltration of the reticuloendothelial system. Foam cells in Niemann-Pick become engorged with sphingomyelin and, to a lesser extent, other membrane lipids including cholesterol. Niemann-Pick is caused by inactivation of the enzyme sphingomyelinase in Types A and B disease, with 27-fold more residual enzyme activity in Type B (see Kolodny et al., 1998, Id.). The pathophysiology of major organ systems in Niemann-Pick can be briefly summarized as follows. The spleen is the most extensively involved organ of Type A and B patients. The lungs are involved to a variable extent, and lung pathology in Type B patients is the major cause of mortality due to chronic bronchopneumonia. Liver involvement is variable, but severely affected patients may have life-threatening cirrhosis, portal hypertension, and ascites. The involvement of the lymph nodes is variable depending on the severity of disease. Central nervous system (CNS) involvement differentiates the major types of Niemann-Pick. While most Type B patients do not experience CNS involvement, it is characteristic in Type A patients. The kidneys are only moderately involved in Niemann Pick disease.
The mucopolysaccharidoses (MPS) comprise a group of LSDs caused by deficiency of enzymes which catalyze the degradation of specific glycosaminoglycans (mucopolysaccharides or GAGs) known as dermatan sulfate and heparan sulfate. GAGs contain long unbranched polysaccharides characterized by a repeating disaccharide unit and are found in the body linked to core proteins to form proteoglycans. Proteoglycans are located primarily in the extracellular matrix and on the surface of cells where they lubricate joints and contribute to structural integrity (see generally Neufeld et al., 1995, The Mucopolysaccharidoses, In: The Metabolic and Molecular Bases of Inherited Diseases, Scriver et al., eds., McGraw-Hill, New York, 7th ed., pages 2465-2494).
The several mucopolysaccharidoses are distinguished by the particular enzyme affected in GAG degradation. For example, MPS I (Hurler-Scheie) is caused by a deficiency of α-L-iduronidase which hydrolyzes the terminal α-L-iduronic acid residues of dermatan sulfate. Symptoms in MPS I vary along a clinical continuum from mild (MPS IS or Scheie disease) to intermediate (MPS IHS or Hurler-Scheie disease) to severe (MPS IH or Hurler disease), and the clinical presentation correlates with the degree of residual enzyme activity. The mean age at diagnosis for Hurler syndrome is about nine months, and the first presenting symptoms are often among the following: coarse facial features, skeletal abnormalities, clumsiness, stiffness, infections and hernias (Cleary M A and Wraith J E, 1995, The Presenting Features of Mucopolysaccharidosis Type IH (Hurler Syndrome), Acta. Paediatr. 84, 337-339; Colville G A and Bax M A, 1996, Early Presentation in the Mucopolysaccharide Disorders, Child: Care, Health and Development 22, 31-36).
Other examples of mucopolysaccharidoses include Hunter (MPS II or iduronate sulfatase deficiency), Morquio (MPS IV; deficiency of galactosamine-6-sulfatase and β-galactosidase in types A and B, respectively) and Maroteaux-Lamy (MPS VI or arylsulfatase B deficiency) (see Neufeld et al., 1995, Id.; Kolodny et al., 1998, Id.).
Pompe disease (also known as glycogen storage disease type II, acid maltase deficiency and glycogenosis type II) is an autosomal recessive LSD characterized by a deficiency of α-glucosidase (also known as acid α-glucosidase and acid maltase). The enzyme α-glucosidase normally participates in the degradation of glycogen to glucose in lysosomes; it can also degrade maltose (see generally Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid α-Glucosidase (Acid Maltase) Deficiency, In: The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th ed., pages 2443-2464). The three recognized clinical forms of Pompe disease (infantile, juvenile and adult) are correlated with the level of residual α-glucosidase activity (Reuser A J et al., 1995, Glycogenosis Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement 3, S61-S69).
Infantile Pompe disease (type I or A) is most common and most severe; characterized by failure to thrive, generalized hypotonia, cardiac hypertrophy, and cardiorespiratory failure within the second year of life. Juvenile Pompe disease (type II or B) is intermediate in severity and is characterized by a predominance of muscular symptoms without cardiomegaly. Juvenile Pompe individuals usually die before reaching 20 years of age due to respiratory failure. Adult Pompe disease (type III or C) often presents as a slowly progressive myopathy in the teenage years or as late as the sixth decade (Felice K J et al., 1995, Clinical Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of the Literature, Medicine 74, 131-135).
In Pompe, it has been shown that α-glucosidase is extensively modified post-translationally by glycosylation, phosphorylation, and proteolytic processing. Conversion of the 110 kilodalton (kDa) precursor to 76 and 70 kDa mature forms by proteolysis in the lysosome is required for optimum glycogen catalysis.
2.2 Therapies for Lysosomal Storage Diseases
Several approaches are being used or pursued for the treatment of LSDs, most of which focus on gene therapy or enzyme replacement therapy for use alone in disease management. Additionally, researchers have identified a number of small molecules for use alone in the management of LSDs. Other, disease-specific approaches, are also under consideration.
Gene Therapy
Replacement of the defective enzyme in a patient with Fabry Disease is considered feasible using a recombinant retrovirus carrying the cDNA encoding α-Gal A to transfect skin fibroblasts obtained from Fabry patients (Medin J A et al., 1996, Correction in Trans for Fabry Disease: Expression, Secretion, and Uptake of α-Galactosidase A in Patient-Derived Cells Driven by a High-Titer Recombinant Retroviral Vector, Proc. Natl. Acad. Sci. USA 93, 7917-7922).
In vitro studies have also suggested that gene therapy may be feasible in Pompe disease. Vectors are being developed from both recombinant retrovirus and recombinant adenovirus (Zaretsky J Z et al., 1997, Retroviral Transfer of Acid α-Glucosidase cDNA to Enzyme-Deficient Myoblasts Results in Phenotypic Spread of the Genotypic Correction by Both Secretion and Fusion, Human Gene Therapy 8, 1555-1563; Pauly D F et al., 1998, Complete Correction of Acid α-Glucosidase Deficiency in Pompe Disease Fibroblasts in Vitro, and Lysosomally Targeted Expression in Neonatal Rat Cardiac and Skeletal Muscle, Gene Therapy 5, 473-480).
Additionally, transfer and expression of the normal α-L-iduronidase gene into autologous bone marrow by retroviral gene transfer has also been demonstrated in non-clinical studies of Hurler Syndrome (Fairbairn et al., 1996, Long-Term in vitro Correction of α-L-Iduronidase Deficiency (Hurler Syndrome) in Human Bone Marrow, Proc. Natl. Acad. Sci. U.S.A. 93, 2025-2030).
Enzyme Replacement Therapy
Enzyme replacement therapy involves administration, preferably intravenous, of an exogenously-produced natural or recombinant enzyme. Enzyme replacement therapy proof-of-principle has been established in a Hurler animal model (Shull R M et al., 1994, Enzyme Replacement in a Canine Model of Hurler Syndrome, Proc. Natl. Acad. Sci. USA 91, 12937-12941). Others have developed effective methods for cell culture expression of recombinant enzyme in sufficient quantities to be collected for therapeutic use (Kakkis E D et al., 1994, Overexpression of the Human Lysosomal Enzyme α-L-Iduronidase in Chinese Hamster Ovary Cells, Prot. Express. Purif. 5, 225-232). However, one unsolved problem is the development of antibodies against the replacement enzyme after long term therapy (Kakkis E D et al., 1996, Long-Term and High-Dose Trials of Enzyme Replacement Therapy in the Canine Model of Mucopolysaccharidosis I, Biochem. Molec. Med. 58, 156-167).
The use of enzyme replacement therapy has also been investigated for patients with Pompe disease. However, effective enzyme replacement therapy requires the use of a precursor α-glucosidase molecule for correct targeting to lysosomes (Van Der Ploeg A T et al., 1987, Breakdown of Lysosomal Glycogen in Cultured Fibroblasts from Glycogenosis Type II Patients After Uptake of Acid α-Glucosidase, J. Neurolog. Sci. 79, 327-336; Van Der Ploeg, A T et al., 1991, Intravenous Administration of Phosphorylated Acid α-Glucosidase Leads to Uptake of Enzyme in Heart and Skeletal Muscle of Mice, J. Clin. Invest. 87, 513-518; Van Der Ploeg A T et al., 1988, Prospect for Enzyme Replacement Therapy in Glycogenosis II Variants: A study on Cultured Muscle Cells, J. Neurol. 235, 392-396; Van Der Ploeg A T et al., 1988, Receptor-Mediated Uptake of Acid α-Glucosidase Corrects Lysosomal Glycogen Storage in Cultured Skeletal Muscle, Pediatr. Res. 24, 90-94). Despite the requirement for a robust production method for human recombinant α-glucosidase, animal and in vitro studies have provided reason for optimism (Van Hove J L K et al., 1996, High-Level Production of Recombination Human Lysosomal Acid α-Glucosidase in Chinese Hamster Ovary Cells Which Targets to Heart Muscle and Corrects Glycogen Accumulation in Fibroblasts from Patients with Pompe Disease, Proc. Natl. Acad. Sci. USA 93, 65-70; Kikuchi T et al., 1998, Clinical and Metabolic Correction of Pompe Disease by Enzyme Therapy in Acid Maltase-Deficient Quail, J. Clin. Invest. 101, 827-833).
Small Molecule Therapy
Recently, a variety of studies have been conducted using several small molecules for storage disease therapy. One class of molecules inhibits upstream generation of lysosomal hydrolase substrate to relieve the input burden to the defective enzyme. This approach has been dubbed “substrate deprivation” therapy. One example of this class of molecules is N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of the ceramide-specific glucosyltransferase (i.e. glucosylceramide synthase) which catalyzes the first step in the synthesis of glycosphingolipids (GSLs). NB-DNJ has been tested in mouse models of Sandhoff disease (Jeyakumar et al., 1999, Proc. Natl. Acad. Sci. USA 96, 6388-6393), Tay-Sachs disease (Platt et al., 1997, Science 276, 428-431), as well as in humans with Gaucher's disease (Cox et al., 2000, Lancet 355, 1481-1485), resulting in an amelioration of symptoms in each of these diseases. A variety of deoxynojirimycin (DNJ) derivatives have also been synthesized as research tools intended for the selective inhibition of the non-lysosomal glucosylceramidase at concentrations in which glucosylceramide synthase and other enzymes are not affected (Overkleeft et al., 1998, J. Biol. Chem. 273, 26522-26527). Certain uses of glucosylceramide synthase inhibitors of the DNJ type either alone (WO 00/62780) or in combination with a glycolipid degrading enzyme (WO 00/62779) have been described.
Another example of the substrate deprivation class of molecules are the amino ceramide-like small molecules which have been developed for glucosylceramide synthase inhibition. Glucosylceramide synthase catalyzes the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. Glucosylceramide itself is the precursor of hundreds of different glycosphingolipids. Amino ceramide-like compounds have been developed for use in Fabry disease (Abe et al., 2000, J. Clin. Invest. 105, 1563-1571; Abe et al., 2000, Kidney Int'l 57, 446-454) and Gaucher's disease (Shayman et al., 2000, Meth. Enzymol. 31, 373-387; U.S. Pat. Nos. 5,916,911; 5,945,442; 5,952,370; 6,030,995; 6,040,332 and 6,051,598). A variety of amino ceramide-like analogues have been synthesized as improved inhibitors of glucosylceramide synthase (see e.g. Lee et al., 1999, J. Biol. Chem. 274, 14662-14669).
Aminoglycosides such as gentamicin and G418 are small molecules which promote read-through of premature stop-codon mutations. These so-called stop-mutation suppressors have been used in Hurler cells to restore a low level of α-L-iduronidase activity (Keeling et al., 2001, Hum. Molec. Genet. 10, 291-299). They have also been developed for use in treating cystic fibrosis individuals having stop mutations (U.S. Pat. No. 5,840,702).
Other Therapies
Various other, disease-specific, treatments have been attempted. For example, a high protein diet in adult Pompe has been suggested to combat muscle wasting, but was effective in improving respiratory or muscle function in only 25% of individuals (Bodamer O A F et al., 1997, Dietary Treatment in Late-Onset Acid Maltase Deficiency, Eur. J. Pediatr. 156, S39-S42). In Hurler disease, bone marrow transplantation has shown limited benefits but carries significant risks (Guffon N et al., 1998, Follow-up of Nine Patients with Hurler Syndrome After Bone Marrow Transplantation, J. Pediatr. 133, 119-125; Gullingsrud E O et al., 1998, Ocular Abnormalities in the Mucopolysaccharidoses After Bone Marrow Transplantation, Ophthalmology 105, 1099-1105; Masterson E L et al., 1996, Hip Dysplasia in Hurler's Syndrome: Orthopaedic Management After Bone Marrow Transplantation, J. Pediatric Orthopaedics 16, 731-733; Peters C et al., 1998, Hurler Syndrome: Past, Present and Future, J. Pediatr. 133, 7-9; Peters C et al., 1998, Hurler Syndrome: II. Outcome of HLA-Genotypically Identical Sibling and HLA-Haploidentical: Related Donor Bone Marrow Transplantation in Fifty-Four Children, Blood 91, 2601-2608). Early surgical intervention for nerve compression has been reported to improve hand function in individuals with Hurler disease (Van Heest A E et al., 1998, Surgical Treatment of Carpal Tunnel Syndrome and Trigger Digits in Children with Mucopolysaccharide Storage Disorders, J. Hand Surgery 23A, 236-243).
Kolodny et al. have provided a general overview of several approaches for treatment of LSDs in current use or development, including bone marrow transplantation, enzyme replacement therapy, and gene therapy (Kolodny et al., 1998, Id.). However, a need exists for defined combination therapies that overcome significant limitations associated with each of these treatment modalities when used alone. The present invention meets this need by providing approaches utilizing combinations of two or more of enzyme replacement therapy, gene therapy and small molecule therapy.